Fluorescent dyes have a wide application in analytical chemistry, biochemistry and molecular biology, such as in DNA sequencing and during detection and amplification of nucleic acids. Fluorescent dyes absorb light of a specific wavelength spectrum (“excitation spectrum”) and re-emit energy at a different specific wavelength spectrum (“emission spectrum”). The fluorescent dyes are frequently used as molecular labels attached to DNA and protein probes or other biomolecules.
Multiplex polymerase chain reaction (multiplex PCR) is a PCR technique that enables amplification of two or more products in parallel in a single reaction tube. It is widely used in genotyping applications and different areas of molecular biology, e.g. in research, forensic and diagnostic applications, including human identification and paternity testing and for diagnosis of infectious diseases or chimerism analysis after allogeneic bone marrow transplantation. Multiplex PCR can also be used for qualitative and semi-quantitative gene expression analysis using cDNA as a starting template or in combination with reverse transcription with mRNA as starting material. The nucleic acids analyzed may for example originate from a variety of eukaryotic (human, animal or plant) and prokaryotic (bacterial) or viral sources.
Multiplex PCR is for example used for the simultaneous detection of multiple marker genes and/or their polymorphisms, e.g. single nucleotide polymorphisms (SNPs), short tandem repeats (STRs) or deletion insertion polymorphisms (DIPs or Indels). Detection of the amplification products and their genotyping is usually carried out by multiple colour fluorescence detection after electrophorectic separation (e.g. capillary gel electrophoresis) in DNA sequencers. In real-time, quantitative multiplex PCR, the amplification of multiple target sequences can be monitored at the same time by simultaneous detection of fluorescence of different fluorescent dyes.
Instruments for multiple color fluorescence detection are often designed and calibrated for the combinations of very specific fluorescent dyes, i.e. in terms of excitation and detection spectra and filter sets.
Many DNA sequence analyzers employ a capillary electrophoresis device in combination with a special multiwavelength fluorescence detection system. The basic setup of such instruments was described by Karger et al. (1991) (Karger A, Harris J M, Gesteland R F. Multiwavelength fluorescence detection for DNA sequencing using capillary electrophoresis. Nucleic Acids Res 18, 4955-4962, 1991) and has been put commercially into practice by the ABI Prism® Genetic Analyzer series (310, 3100, 3130 etc.) of Applied Biosystems (Foster City, Calif., USA). These instruments can by used for DNA sequence analysis, multiplex genotyping of genetic polymorphisms (SNPs, DIPs, STRs), mRNA expression analysis or, more generally, for amplified DNA fragment length analysis. The latter is also the current standard for multiplex-PCR STR genotyping in the field of forensics and paternity testing. In this case at least one primer of each primer pair applied during a multiplex amplification is labeled by a covalently bond fluorescent dye at its 5′-OH end or at an internal base. The optical detection units of these instruments use a multiline argon ion laser, adjustable to 10 mW, which excites multiple fluorophores at 488 nm (main wavelength) and 514 nm (Wenz H. Robertson J M, Menchen S, Oaks F, Demorest D M, Scheibler D, Rosenblum B B, Wike C, Gilbert D A, Efcavitch J W. High-precision genotyping by denaturing capillary electrophoresis. Genome Res 8, pp 69-80, 1998). Fluorescence emission is recorded between 525 and 680 nm. The emitted light is separated for simultaneous multiple color fluorescence detection by a spectrograph (prism) and imaged on a cooled CCD (charge coupled device) camera. In the current configurations up to 5 wavelength bands can be defined on the CCD chip by digital band pass filters of app. 10 nm weight. The relative position of these digital band pass filters is proprietarily specified by the manufacturer for a restricted number of dye combinations (see table 1). These settings of the device are also referred to as virtual filter sets. The accurate position and range of the digital band pass filters is not published. The new generation of multi-capillary DNA sequencer such as ABI Prism® 3130 Genetic Analyzer possess in addition also two variable virtual filtersets, termed “any4dyes” or “any5dyes”, to allow the customers to adjust preset digital band pass filters for other dye combinations. For this purpose so called condition bound numbers can by varied to some extend. However again, there is no information about the relative position and wavelength range of these digital band pass filters.
TABLE 1Known fluorescent dye combinations and virtual filtersets for ABI Prism ® Genetic Analyzers.Fluorescence dye combinations and colour canalsOrangeRecommendedBlueGreenYellowRed(far red)virtual filterset6-FAMJOETAMRAROX—F or A6-FAMTETHEXTAMRA—C5, 6-FAMJOETAMRAROX—F or A6-FAMJOENEDROX—F6-FAMHEXNEDROX—F or D6-FAMVICNEDPETLIZG55, 6-FAM: 5- and 6-carboxyfluorescein;6-FAM: 6-carboxyfluorescein;JOE: 6-carboxy-4′-, 5′-dichloro-2′-, 7′-dimethoxy-fluorescein;HEX: 4,7,2′,4′,5′,7′-hexachloro-6-carboxy-fluorescein;TET: 4,7,2′,7′-tetrachloro-6-carboxy-fluorescein;TAMRA: 6-carboxytetramethyl-rhodamine;VIC: 2′-chloro-7′phenyl-1,4-dichloro-6-carboxy-fluorescein;NED: 2′-chloro-5′-fluoro-7′,8′-benzo-1,4-dichloro-6-carboxyfluorescein;ROX: 5- and 6-carboxy-X-rhodamin;PET and LIZ are unpublished proprietary dyes of Applied Biosystems (Foster City, CA, USA).
Furthermore, a spectral calibration must be performed for a selected multi-colour filter set for simultaneous detection of 4 or 5 different colours to generate a mathematical matrix for the correction of overlapping fluorescence emission spectra of the dyes. Mathematical algorithms underlying this calculation are not published.
It is also important to note that the optical setup of ABI Prism® Genetic Analyzers for simultaneous detection of different fluorescent dyes which consists of a single argon ion laser, a spectrograph, virtual filtersets in combination with a CCD-camera and spectral calibration algorithms fundamentally differs from other sequencers or from those used by real-time PCR thermocyclers. The latter possess sets of excitation and emission filters which analyze different dyes by distinct filter combinations and short time offsets (time resolved). Furthermore, the specific optical setup of ABI Prism®Genetic Analyzers also limits the compatibility of different dyes for multi-fluorescence detection.
As outlined in table 1 there are only a few sets of fluorescence dyes available which can be used for DNA oligonucleotide labelling and simultaneous detection by DNA sequencing automates of Applied Biosystems. The relative sensitivity of these dyes dramatically declines between the blue and red colour canal due to the use of a single argon laser which mostly excites at 488 nm. In addition, the relative sensitivity and proper analytical separation of the colours is influenced by spectral overlaps. Further restrictions arise from the fact that many of these dyes (e.g. VIC, HEX, NED, PET, LIZ) are proprietary of Applied Biosystems. Customized PCR primers labelled with the dyes VIC, NED and PET are only available via the synthesis laboratory of this company, and LIZ is only available in combination with a labelled DNA length standard as a commercial product from Applied Biosystems. Furthermore, the chemical structures of PET and LIZ are not known. Thus, there is a need for new colour combinations for technical and economical reasons.